Methoxyethoxy oligonucleotides for modulation of protein kinase C expression

ABSTRACT

Compositions and methods are provided for modulating the expression of protein kinase C. Oligonucleotides are provided which are targeted to nucleic acids encoding PKC. The oligonucleotides contain a methoxyethoxy (--O--CH 2  CH 2  OCH 3 ) modification at the 2&#39; position of at least one nucleotide. Methods of inhibiting PKC expression and methods of treating conditions associated with expression of PKC using oligonucleotides of the invention are disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 08/478,178, filed Jun. 7, 1995, now U.S. Pat. No. 5,882,927,which is a continuation-in-part of U.S. patent application Ser. No.08/089,996, filed Jul. 9, 1993, now U.S. Pat. No. 5,703,054, which inturn is a continuation-in-part of U.S. patent application Ser. No.07/852,852 filed Mar. 16, 1992, now abandoned.

FIELD OF THE INVENTION

This invention relates to compositions and methods for modulation of theexpression of protein kinase C. In particular, this invention relates toantisense oligonucleotides specifically hybridizable with nucleic acidsencoding protein kinase C. These oligonucleotides have been found tomodulate the expression of protein kinase C. These compositions andmethods can be used diagnostically or therapeutically.

BACKGROUND OF THE INVENTION

The phosphorylation of proteins plays a key role in the transduction ofextracellular signals into the cell. The enzymes, called kinases, whicheffect such phosphorylations are targets for the action of growthfactors, hormones, and other agents involved in cellular metabolism,proliferation and differentiation. One of the major signal transductionpathways involves the enzyme protein kinase C (PKC), which is known tohave a critical influence on cell proliferation and differentiation. PKCis activated by diacylglycerols (DAGs), which are metabolites releasedin signal transduction.

Interest in PKC was stimulated by the finding that PKC is the major, andperhaps only, cellular receptor through which a class of tumor-promotingagents called phorbol esters exert their pleiotropic effects on cells(Gescher et al., Anti-Cancer Drug Design 4:93-105 (1989)). Phorbolscapable of tumor production can mimic the effect of DAG in activatingPKC, suggesting that these tumor promoters act through PKC and thatactivation of this enzyme is at least partially responsible for theresulting tumorigenesis (Parker et al., Science 233:853-866 (1986)).

Experimental evidence indicates that PKC plays a role in growth controlin colon cancer. It is believed that specific bacteria in the intestinaltract convert lipids to DAG, thus activating PKC and altering cellproliferation. This may explain the correlation between high dietary fatand colon cancer (Weinstein, Cancer Res. (Suppl.) 51:5080s-5085s(1991)). It has also been demonstrated that a greater proportion of thePKC in the colonic mucosa of patients with colorectal cancer is in anactivated state compared to that of patients without cancer (Sakanoue etal., Int. J. Cancer 48:803-806 (1991)).

Increased tumorigenicity is also correlated with overexpression of PKCin cultured cells inoculated into nude mice. A mutant form of PKCinduces highly malignant tumor cells with increased metastaticpotential. Sphingosine and related inhibitors of PKC activity have beenshown to inhibit tumor cell growth and radiation-induced transformationin vivo (Endo et al., Cancer Research 51:1613-1618 (1991); Borek et al.,Proc. Natl. Acad. Sci. 88:1953-1957 (1991)). A number of experimental orclinically useful anti-cancer drugs show modulatory effects on PKC.Therefore, inhibitors of PKC may be important cancer-preventive ortherapeutic agents. PKC has been suggested as a plausible target formore rational design of conventional anti-cancer drugs (Gescher, A. andDale, I. L., Anti-Cancer Drug Design, 4:93-105 (1989)).

Experiments also indicate that PKC plays an important role in thepathophysiology of hyperproliferative skin disorders such as psoriasisand skin cancer. Psoriasis is characterized by inflammation,hyperproliferation of the epidermis and decreased differentiation ofcells. Various studies indicate a role for PKC in causing thesesymptoms. PKC stimulation in cultured keratinocytes can be shown tocause hyperproliferation. Inflammation can be induced by phorbol estersand is regulated by PKC. DAG is implicated in the involvement of PKC indermatological diseases, and is formed to an increased extent inpsoriatic lesions.

Inhibitors of PKC have been shown to have both antiproliferative andantiinflammatory effects in vitro. Some antipsoriasis drugs, such ascyclosporine A and anthralin, have been shown to inhibit PKC. Inhibitionof PKC has been suggested as a therapeutic approach to the treatment ofpsoriasis (Hegemann, L. and G. Mahrle, Pharmacology of the Skin, H.Mukhtar, ed., p. 357-368, CRC Press, Boca Raton, Fla., 1992).

The oligonucleotides of the invention are believed to be useful in thetherapeutic treatment of diseases associated with PKC. Such diseasesinclude hyperproliferative and inflammatory conditions includingpsoriasis, tumors and cancers, for example glioblastoma, bladder cancer,skin cancer, breast cancer, lung cancer and colon cancer.

PKC is not a single enzyme, but a family of enzymes. At the present timeat least seven isoforms (isozymes) of PKC have been identified: isoformsα, β, and γ have been purified to homogeneity, and isoforms δ, ε, ζ andη have been identified by molecular cloning. These isozymes havedistinct patterns of tissue and organ localization (see Nishizuka,Nature, 334:661-665 (1988) for review) and may serve differentphysiological functions.

It is presently believed that different PKC isozymes may be involved invarious disease processes depending on the organ or tissue in which theyare expressed. For example, in psoriatic lesions there is an alterationin the ratio between PKC-α and PKC-β, with preferential loss of PKC-βcompared to normal skin (Hegemann, L. and G. Mahrle, Pharmacology of theSkin, H. Mukhtar, ed., p. 357-368, CRC Press, Boca Raton, Fla. 1992).

Although numerous compounds have been identified as PKC inhibitors (seeHidaka and Hagiwara, Trends in Pharm. Sci. 8:162-164 (1987) for review),few have been found which inhibit PKC specifically. While the quinolinesulfonamide derivatives such as1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) inhibit PKC atmicromolar concentrations, they exhibit similar enzyme inhibitionkinetics for PKC and the CAMP-dependent and cGMP-dependent proteinkinases. Staurosporine, an alkaloid product of Streptomyces sp., and itsanalogs, are the most potent in vitro inhibitors of PKC identified todate. However, they exhibit only limited selectivity among differentprotein kinases (Gescher, Anti-Cancer Drug Design 4:93-105 (1989)).Certain ceramides and sphingosine derivatives have been shown to havePKC inhibitory activity and to have promise for therapeutic uses,however, there remains a long-felt need for specific inhibitors of theenzymes.

There is also a desire to inhibit specific PKC isozymes, both as aresearch tool and in diagnosis and treatment of diseases which may beassociated with particular isozymes. Godson et al. (J. Biol. Chem.268:11946-11950 (1993)) disclosed use of stable transfection ofantisense PKC-α cDNA in cytomegalovirus promotor-based expressionvectors to specifically decrease expression of PKC-α protein byapproximately 70%. It was demonstrated that this inhibition caused aloss of phospholipase A₂ -mediated arachidonic acid release in responseto the phorbol ester PMA. Attempts by the same researchers at inhibitingPKC activity with oligodeoxynucleotides were ultimately unsuccessful dueto degradation of oligonucleotides. Ahmad et al. disclose thattransfection of the human glioblastoma cell line, U-87, with vectorsexpressing antisense RNA to PKCα inhibits growth of the glioblastomacells in vitro and in vivo (Ahmad et al., Neurosurg. 35:904-908 (1994)).Diaz-Meco Conde et al. disclose a peptide corresponding to thepseudo-substrate region of PKC-ζ and oligonucleotides antisense to thisisozyme (Wo Application 93/20101). Alvaro et al. have identified a novelmutant form of PKC associated with tumors and disclose oligonucleotidesequences complementary to the mutant form (WO Application 94/29455).

SUMMARY OF THE INVENTION

In accordance with the present invention, oligonucleotides are providedthat are specifically hybridizable with a nucleic acid that encodes PKCαand are capable of inhibiting PKCα expression. This relationship iscommonly denominated as "antisense". The oligonucleotides contain amethoxyethoxy (--O--CH₂ CH₂ OCH₃) modification at the 2' position of thesugar moiety of at least one nucleotide. These oligonucleotides,referred to herein as "2'-methoxyethoxy" or "2'-O--CH₂ CH₂ OCH₃ "compounds, have been found to be surprisingly more potent thanpreviously tested oligonucleotides for inhibiting PKC expression. Theyare believed to be useful both diagnostically and therapeutically, andare believed to be particularly useful in the methods of the presentinvention.

Also provided are methods for modulating the expression of PKCα usingthe oligonucleotides of the invention. These methods are believed to beuseful both therapeutically and diagnostically as a consequence of therelationship between PKCα and inflammation and hyperproliferation.

Other aspects of the invention are directed to methods for diagnosticsand treatment of conditions associated with PKCα.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B are a set of line graphs showing the effect of2'-methoxyethoxy modified oligonucleotides having SEQ ID NO: 1 on PKCαmRNA levels in A549 cells. FIG. 1A shows the effect of ISIS 9605compared to the deoxyphosphorothioate compound, ISIS 3521. FIG. 1B showsthe effect of ISIS 9606 compared to the deoxyphosphorothioate compound,ISIS 3521.

FIG. 2 is a line graph showing anti-tumor activity of ISIS 3521 on A549tumor growth in nude mice. Each dashed line represents tumor volume inone animal treated with control oligonucleotide; each solid linerepresents tumor volume in one animal treated with ISIS 3521.

FIG. 3 is a line graph showing the effect of ISIS 3521 on growth ofhuman MDA-MB231 tumors in nude mice. Each line represents tumor volumein one animal. =control; ∘=oligonucleotide at 60 mg/kg;Δ=oligonucleotide at 6 mg/kg.

FIG. 4 is a line graph showing a "crossover" experiment to evaluate theeffect of ISIS 3521 on U-87 glioblastoma cells in nude mice in vivo. Theexperiment was carried out with oligonucleotide doses of 2 mg/kg and 20mg/kg and then treatment was switched (arrow). The group which hadoriginally received ISIS 3521 at 20 mg/kg ("high dose-to-control" group,closed triangles) then received saline and the group which hadoriginally received ISIS 3521 at 2 mg/kg ("low dose-to-high dose", opentriangles) then received ISIS 3521 at 20 mg/kg. S=sense oligonucleotide(control); AS=antisense oligonucleotide (ISIS 3521) targeted to PKCα.

FIGS. 5A and 5E are line graphs showing the effects of once dailytreatment with oligonucleotides having SEQ ID NO: 1 on growth of humancolon carcinoma (Colo 205) tumor xenografts subcutaneously transplantedin female Balb/c nude mice. FIG. 5A shows the effect of thedeoxyphosphorothioate compound, ISIS 3521. FIG. 5B shows the effect ofthe 2'-methoxyethoxy modified compound, ISIS 12723.

DETAILED DESCRIPTION OF THE INVENTION

Oligonucleotides have been employed as therapeutic moieties for thetreatment of disease states in animals and man. For example, workers inthe field have now identified antisense, triplex and otheroligonucleotide compositions which are capable of modulating expressionof genes implicated in viral, fungal and metabolic diseases. A number ofoligonucleotides are presently in clinical trials for a variety ofindications including viral infections and inflammatory conditions.

Current agents which modulate the activity or metabolism of proteinkinase C exhibit many unacceptable side effects due to their lack ofspecificity, or they exhibit only limited effectiveness in inhibitingthe enzyme. The instant invention circumvents problems encountered byprior workers by modulating the production of the enzyme, rather thaninhibiting the enzyme directly, to achieve the therapeutic effect. Inthe instant invention, the oligonucleotide is designed to bind directlyto mRNA or to a gene, ultimately modulating the amount of PKC proteinmade from the gene.

This relationship between an oligonucleotide and its complementarynucleic acid target to which it hybridizes is commonly referred to as"antisense". "Targeting" an oligonucleotide to a chosen nucleic acidtarget, in the context of this invention, is a multistep process. Theprocess usually begins with identifying a nucleic acid sequence whosefunction is to be modulated. This may be, as examples, a cellular gene(or mRNA made from the gene) whose expression is associated with aparticular disease state, or a foreign nucleic acid from an infectiousagent. In the present invention, the target is a nucleic acid encodingPKC; in other words, a PKC gene or mRNA expressed from a PKC gene. Thetargeting process also includes determination of a site or sites withinthe nucleic acid sequence for the oligonucleotide interaction to occursuch that the desired effect--modulation of gene expression--willresult. Once the target site or sites have been identified,oligonucleotides are chosen which are sufficiently complementary to thetarget, i.e., hybridize sufficiently well and with sufficientspecificity, to give the desired modulation.

In the context of this invention "modulation" means either inhibition orstimulation. Inhibition of PKC gene expression is presently thepreferred form of modulation. This modulation can be measured in wayswhich are routine in the art, for example by Northern blot assay of mRNAexpression or Western blot assay of protein expression as taught in theexamples of the instant application. Effects on cell proliferation ortumor cell growth can also be measured, as taught in the examples of theinstant application.

"Hybridization", in the context of this invention, means hydrogenbonding, also known as Watson-Crick base pairing, between complementarybases, usually on opposite nucleic acid strands or two regions of anucleic acid strand. Guanine and cytosine are examples of complementarybases which are known to form three hydrogen bonds between them. Adenineand thymine are examples of complementary bases which form two hydrogenbonds between them.

"Specifically hybridizable" and "complementary" are terms which are usedto indicate a sufficient degree of complementarity such that stable andspecific binding occurs between the DNA or RNA target and theoligonucleotide.

It is understood that an oligonucleotide need not be 100% complementaryto its target nucleic acid sequence to be specifically hybridizable. Anoligonucleotide is specifically hybridizable when binding of theoligonucleotide to the target interferes with the normal function of thetarget molecule to cause a loss of utility, and there is a sufficientdegree of complementarity to avoid non-specific binding of theoligonucleotide to non-target sequences under conditions in whichspecific binding is desired, i.e., under physiological conditions in thecase of in vivo assays or therapeutic treatment or, in the case of invitro assays, under conditions in which the assays are conducted.

In the context of this invention, the term "oligonucleotide" refers toan oligomer or polymer of nucleotide or nucleoside monomers consistingof naturally occurring bases, sugars and intersugar (backbone) linkages.The term "oligonucleotide" also includes oligomers comprisingnon-naturally occurring monomers, or portions thereof, which functionsimilarly. Modifications may be on one or more bases, sugars, orbackbone linkages, or combinations of these; such modifications are wellknown in the art. Modified or substituted oligonucleotides are oftenpreferred over native forms because of properties such as, for example,enhanced cellular uptake and increased stability in the presence ofnucleases.

The oligonucleotides may be chimeric oligonucleotides. "Chimericoligonucleotides" or "chimeras", in the context of this invention, areoligonucleotides which contain two or more chemically distinct regions,each made up of at least one nucleotide. These oligonucleotidestypically contain at least one region of modified nucleotides thatconfers one or more beneficial properties (such as, for example,increased nuclease resistance, increased uptake into cells, increasedbinding affinity for the RNA target) and a region that is a substratefor RNase H cleavage. In one embodiment, a chimeric oligonucleotidecomprises at least one region modified to increase target bindingaffinity, and, usually, a region that acts as a substrate for RNAse H.Affinity of an oligonucleotide for its target (in this case a nucleicacid encoding PKC) is routinely determined by measuring the Tm of anoligonucleotide/target pair, which is the temperature at which theoligonucleotide and target dissociate; dissociation is detectedspectrophotometrically. The higher the Tm, the greater the affinity ofthe oligonucleotide for the target. Such modifications are routinelyincorporated into oligonucleotides and these oligonucleotides have beenshown to have a higher Tm (i.e., higher target binding affinity) than2'-deoxyoligonucleotides against a given target. The effect of suchincreased affinity is to greatly enhance antisense oligonucleotideinhibition of PKC gene expression. RNAse H is a cellular endonucleasethat cleaves the RNA strand of RNA:DNA duplexes; activation of thisenzyme therefore results in cleavage of the RNA target, and thus cangreatly enhance the efficiency of antisense inhibition. Cleavage of theRNA target can be routinely demonstrated by gel electrophoresis. Inanother embodiment, the chimeric oligonucleotide is also modified toenhance nuclease resistance. Cells contain a variety of exo- andendo-nucleases which can degrade nucleic acids. A number of nucleotideand nucleoside modifications have been shown to make the oligonucleotideinto which they are incorporated more resistant to nuclease digestionthan the native oligodeoxynucleotide. Nuclease resistance is routinelymeasured by incubating oligonucleotides with cellular extracts orisolated nuclease solutions and measuring the extent of intactoligonucleotide remaining over time, usually by gel electrophoresis.Oligonucleotides which have been modified to enhance their nucleaseresistance survive intact for a longer time than unmodifiedoligonucleotides. A variety of oligonucleotide modifications have beendemonstrated to enhance or confer nuclease resistance. In some cases,oligonucleotide modifications which enhance target binding affinity arealso, independently, able to enhance nuclease resistance.

The oligonucleotides of the present invention contain a methoxyethoxy(--O--CH₂ CH₂ OCH₃) modification at the 2' position of the sugar moietyof at least one nucleotide. This modification has been shown to increaseboth affinity of the oligonucleotide for its target and nucleaseresistance of the oligonucleotide. Oligonucleotides in accordance withthis invention can comprise a plurality of nucleotide subunits whicheach further comprise a methoxyethoxy (--O--CH₂ CH₂ OCH₃) modificationat the 2' position of their sugar moiety. Thus, only one, a plurality,or all of the nucleotide subunits of the olignucleotides of theinvention can comprise a methoxyethoxy (--O--CH₂ CH₂ OCH₃) modificationat the 2' position of the sugar moiety. Oligonucleotides comprising aplurality of nucleotide subunits having a 2'-methoxyethoxy modificationcan have such a modification on any of the nucleotide subunits withinthe oligonucleotide. Oligonucleotides in accordance with this inventionare preferably from about 8 to about 50 nucleotides in length. In thecontext of this invention it is understood that this encompassesnon-naturally occurring oligomers as hereinbefore described, having 8 to50 monomers.

The oligonucleotides used in accordance with this invention may beconveniently and routinely made through the well-known technique ofsolid phase synthesis (Martin, P., Helv. Chim. Acta, 1995, 78, 486-504).Equipment for such synthesis is sold by several vendors includingApplied Biosystems. Any other means for such synthesis may also beemployed; the actual synthesis of the oligonucleotides is well withinthe talents of the routineer. It is also well known to use similartechniques to prepare other oligonucleotides such as thephosphorothioates and alkylated derivatives. It is also well known touse similar techniques and commercially available modified amidites andcontrolled-pore glass (CPG) products such as biotin, fluorescein,acridine or psoralen-modified amidites and/or CPG (available from GlenResearch, Sterling Va.) to synthesize fluorescently labeled,biotinylated or other conjugated oligonucleotides. The intersugarlinkages between the individual nucleotides of the oligonucleotides ofthe invention can be, but are not limited to, all phosphodiesterlinkages, all phosphorothioate linkages, or a mixture of bothphosphodiester and phosphorothioate linkages. In addition, theoligonucleotides of the invention can comprise other intersugar linkagesas known to the skilled artisan.

In accordance with this invention, persons of ordinary skill in the artwill understand that messenger RNA includes not only the information toencode a protein using the three letter genetic code, but alsoassociated ribonucleotides which form a region known to such persons asthe 5'-untranslated region, the 3'-untranslated region, the 5' capregion and intron/exon junction ribonucleotides. Thus, oligonucleotidesmay be formulated in accordance with this invention which are targetedwholly or in part to these associated ribonucleotides as well as to theinformational ribonucleotides. In preferred embodiments, theoligonucleotide is specifically hybridizable with a transcriptioninitiation site, a translation initiation site, a 5' cap region, anintron/exon junction, coding sequences or sequences in the 5'- or3'-untranslated region.

The oligonucleotides of this invention are designed to be hybridizablewith messenger RNA derived from the PKC gene. Such hybridization, whenaccomplished, interferes with the normal roles of the messenger RNA tocause a modulation of its function in the cell. The functions ofmessenger RNA to be interfered with may include all vital functions suchas translocation of the RNA to the site for protein translation, actualtranslation of protein from the RNA, splicing of the RNA to yield one ormore mRNA species, and possibly even independent catalytic activitywhich may be engaged in by the RNA. The overall effect of suchinterference with the RNA function is to modulate expression of the PKCgene.

The oligonucleotides of this invention can be used in diagnostics,therapeutics, prophylaxis, and as research reagents and in kits. Sincethe oligonucleotides of this invention hybridize to the PKC gene and itsmRNA, sandwich and other assays can easily be constructed to exploitthis fact. Furthermore, since the oligonucleotides of this inventionhybridize specifically to particular isozymes of the PKC mRNA, suchassays can be devised for screening of cells and tissues for particularPKC isozymes. Such assays can be utilized for diagnosis of diseasesassociated with various PKC forms. Provision of means for detectinghybridization of oligonucleotide with the PKC gene can routinely beaccomplished. Such provision may include enzyme conjugation,radiolabelling or any other suitable detection systems. Kits fordetecting the presence or absence of PKC may also be prepared.

The present invention is also suitable for diagnosing abnormalproliferative states in tissue or other samples from patients suspectedof having a hyperproliferative disease such as cancer or psoriasis. Theability of the oligonucleotides of the present invention to inhibit cellproliferation may be employed to diagnose such states. A number ofassays may be formulated employing the present invention, which assayswill commonly comprise contacting a tissue sample with anoligonucleotide of the invention under conditions selected to permitdetection and, usually, quantitation of such inhibition. In the contextof this invention, to "contact" tissues or cells with an oligonucleotideor oligonucleotides means to add the oligonucleotide(s), usually in aliquid carrier, to a cell suspension or tissue sample, either in vitroor ex vivo, or to administer the oligonucleotide(s) to cells or tissueswithin an animal. Similarly, the present invention can be used todistinguish PKC-associated tumors, particularly tumors associated withPKCα, from tumors having other etiologies, in order that an efficacioustreatment regime can be designed.

The oligonucleotides of this invention may also be used for researchpurposes. Thus, the specific hybridization exhibited by theoligonucleotides may be used for assays, purifications, cellular productpreparations and in other methodologies which may be appreciated bypersons of ordinary skill in the art.

The oligonucleotides of the invention are also useful for detection anddiagnosis of PKC expression, particularly the specific expression ofPKCα. For example, radiolabeled oligonucleotides can be prepared by ³² Plabeling at the 5' end with polynucleotide kinase. Sambrook et al.,Molecular Cloning. A Laboratory Manual, Cold spring Harbor LaboratoryPress, 1989, Volume 2, p. 10.59. Radiolabeled oligonucleotides are thencontacted with tissue or cell samples suspected of PKC expression andthe sample is washed to remove unbound oligonucleotide. Radioactivityremaining in the sample indicates bound oligonucleotide (which in turnindicates the presence of PKC) and can be quantitated using ascintillation counter or other routine means. Radiolabeled oligo canalso be used to perform autoradiography of tissues to determine thelocalization, distribution and quantitation of PKC expression forresearch, diagnostic or therapeutic purposes. In such studies, tissuesections are treated with radiolabeled oligonucleotide and washed asdescribed above, then exposed to photographic emulsion according toroutine autoradiography procedures. The emulsion, when developed, yieldsan image of silver grains over the regions expressing PKC. Quantitationof the silver grains permits PKC expression to be detected.

Analogous assays for fluorescent detection of PKC expression can bedeveloped using oligonucleotides of the invention which are conjugatedwith fluorescein or other fluorescent tag instead of radiolabeling. Suchconjugations are routinely accomplished during solid phase synthesisusing fluorescently labeled amidites or CPG (e.g., fluorescein-labeledamidites and CPG available from Glen Research, Sterling Va. See 1993Catalog of Products for DNA Research, Glen Research, Sterling Va., p.21).

Each of these assay formats is known in the art. One of skill couldeasily adapt these known assays for detection of PKC expression inaccordance with the teachings of the invention providing a novel anduseful means to detect PKC expression, particularly of PKCα.

For therapeutic or prophylactic treatment, oligonucleotides areadministered in accordance with this invention. Oligonucleotides may beformulated in a pharmaceutical composition, which may includepharmaceutically acceptable carriers, thickeners, diluents, buffers,preservatives, surface active agents and the like in addition to theoligonucleotide.

The pharmaceutical composition may be administered in a number of waysdepending on whether local or systemic treatment is desired, and on thearea to be treated. Administration may be done topically (includingophthalmically, vaginally, rectally, intranasally), orally, byinhalation, or parenterally, for example by intravenous drip or byintravenous, subcutaneous, intraperitoneal or intramuscular injection.

Formulations for topical administration may include ointments, lotions,creams, gels, drops, suppositories, sprays, liquids and powders.Conventional pharmaceutical carriers, aqueous, powder or oily bases,thickeners and the like may be necessary or desirable. Coated condomsmay also be useful.

Compositions for oral administration include powders or granules,suspensions or solutions in water or non-aqueous media, capsules,sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers,dispersing aids or binders may be desirable.

Formulations for parenteral administration may include sterile aqueoussolutions which may also contain buffers, diluents and other suitableadditives.

Dosing is dependent on severity and responsiveness of the condition tobe treated, but will normally be one or more doses per day, with courseof treatment lasting from several days to several months or until a cureis effected or a diminution of disease state is achieved. Persons ofordinary skill can easily determine optimum dosages, dosingmethodologies and repetition rates. Optimum dosages may vary dependingon the relative potency of individual oligonucleotides, and cangenerally be calculated based on EC50's in in vitro and in vivo animalstudies. For example, given the molecular weight of compound (derivedfrom oligonucleotide sequence and chemical structure) and an effectivedose such as an IC50, for example (derived experimentally), a dose inmg/kg is routinely calculated.

Thus, in the context of this invention, by "therapeutically effectiveamount" is meant the amount of the compound which is required to have atherapeutic effect on the treated mammal. This amount, which will beapparent to the skilled artisan, will depend upon the type of mammal,the age and weight of the mammal, the type of disease to be treated,perhaps even the gender of the mammal, and other factors which areroutinely taken into consideration when treating a mammal with adisease. A therapeutic effect is assessed in the mammal by measuring theeffect of the compound on the disease state in the animal. For example,if the disease to be treated is psoriasis, a reduction or ablation ofthe skin plaque is an indication that the administered dose has atherapeutic effect. Similarly, in mammals being treated for cancer,therapeutic effects are assessed by measuring the rate of growth or thesize of the tumor, or by measuring the production of compounds such ascytokines, production of which is an indication of the progress orregression of the tumor.

The following examples illustrate the present invention and are notintended to limit the same.

EXAMPLES Example 1

Oligonucleotide synthesis

Unmodified DNA oligonucleotides were synthesized on an automated DNAsynthesizer (Applied Biosystems model 380B) using standardphosphoramidite chemistry with oxidation by iodine.β-cyanoethyldiisopropyl-phosphoramidites were purchased from PerSeptiveBiosystems (Framingham, Mass.). For phosphorothioate oligonucleotides,the standard oxidation bottle was replaced by a 0.2 M solution of3H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrile for the stepwisethiation of the phosphite linkages. The thiation cycle wait step wasincreased to 68 seconds and was followed by the capping step.

After cleavage from the controlled pore glass column (AppliedBiosystems) and deblocking in concentrated ammonium hydroxide at 55° C.for 18 hours, the oligonucleotides were purified by precipitation twiceout of 0.5 M NaCl with 2.5 volumes ethanol. Analytical gelelectrophoresis was accomplished in 20% acrylamide, 8 M urea, 45 mMTris-borate buffer, Ph 7.0.

Phosphorothioate oligonucleotides targeted to human PKCα were designedusing the cDNA sequence published by Finkenzeller et al., Nucl. AcidsRes. 18:2183 (1990); Genbank accession number X52479.

2'-O--CH₂ CH₂ OCH₃ modified oligonucleotides:

Oligonucleotides having 2'-O--CH₂ CH₂ OCH₃ modified nucleotides weresynthesized according to the method of Martin. Helv. Chim. Acta 1995,78,486-504. All 2'-O--CH₂ CH₂ OCH₃₋₋ cytosines were 5-methyl cytosines.

5-Methyl cytosine monomers:

2,2'-Anhydro[1-(β-D-arabinofuranosyl)-5-methyluridine]:

5-Methyluridine (ribosylthymine, commercially available through Yamasa,Choshi, Japan) (72.0 g, 0.279 M), diphenylcarbonate (90.0 g, 0.420 M)and sodium bicarbonate (2.0 g, 0.024 M) were added to DMF (300 mL). Themixture was heated to reflux, with stirring, allowing the evolved carbondioxide gas to be released in a controlled manner. After 1 hour, theslightly darkened solution was concentrated under reduced pressure. Theresulting syrup was poured into diethylether (2.5 L), with stirring. Theproduct formed a gum. The ether was decanted and the residue wasdissolved in a minimum amount of methanol (ca. 400 mL). The solution waspoured into fresh ether (2.5 L) to yield a stiff gum. The ether wasdecanted and the gum was dried in a vacuum oven (60° C. at 1 mm Hg for24 h) to give a solid which was crushed to a light tan powder (57 g, 85%crude yield). The material was used as is for further reactions.

2'-O-Methoxyethyl-5-methyluridine:

2,2'-Anhydro-5-methyluridine (195 g, 0.81 M), tris(2-methoxyethyl)borate(231 g, 0.98 M) and 2-methoxyethanol (1.2 L) were added to a 2 Lstainless steel pressure vessel and placed in a pre-heated oil bath at160° C. After heating for 48 hours at 155-160° C., the vessel was openedand the solution evaporated to dryness and triturated with MeOH (200mL). The residue was suspended in hot acetone (1 L). The insoluble saltswere filtered, washed with acetone (150 mL) and the filtrate evaporated.The residue (280 g) was dissolved in CH₃ CN (600 mL) and evaporated. Asilica gel column (3 kg) was packed in CH₂ Cl₂ /acetone/MeOH (20:5:3)containing 0.5% Et₃ NH. The residue was dissolved in CH₂ Cl₂ (250 mL)and adsorbed onto silica (150 g) prior to loading onto the column. Theproduct was eluted with the packing solvent to give 160 g (63%) ofproduct.

2'-O-Methoxyethyl-5'-O-dimethoxytrityl-5-methyluridine:

2'-O-Methoxyethyl-5-methyluridine (160 g, 0.506 M) was co-evaporatedwith pyridine (250 mL) and the dried residue dissolved in pyridine (1.3L). A first aliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) wasadded and the mixture stirred at room temperature for one hour. A secondaliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) was added and thereaction stirred for an additional one hour. Methanol (170 mL) was thenadded to stop the reaction. HPLC showed the presence of approximately70% product. The solvent was evaporated and triturated with CH₃ CN (200mL). The residue was dissolved in CHCl₃ (1.5 L) and extracted with 2×500mL of saturated NaHCO₃ and 2×500 mL of saturated NaCl. The organic phasewas dried over Na₂ SO₄, filtered and evaporated. 275 g of residue wasobtained. The residue was purified on a 3.5 kg silica gel column, packedand eluted with EtOAc/Hexane/Acetone (5:5:1) containing 0.5% Et₃ NH. Thepure fractions were evaporated to give 164 g of product. Approximately20 g additional was obtained from the impure fractions to give a totalyield of 183 g (57%).

3'-O-Acetyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methyluridine:

2'-O-Methoxyethyl-5'-O-dimethoxytrityl-5-methyluridine (106 g, 0.167 M),DMF/pyridine (750 mL of a 3:1 mixture prepared from 562 mL of DMF and188 mL of pyridine) and acetic anhydride (24.38 mL, 0.258 M) werecombined and stirred at room temperature for 24 hours. The reaction wasmonitored by tlc by first quenching the tlc sample with the addition ofMeOH. Upon completion of the reaction, as judged by tlc, MeOH (50 mL)was added and the mixture evaporated at 35° C. The residue was dissolvedin CHCl₃ (800 mL) and extracted with 2×200 mL of saturated sodiumbicarbonate and 2×200 mL of saturated NaCl. The water layers were backextracted with 200 mL of CHCl₃. The combined organics were dried withsodium sulfate and evaporated to give 122 g of residue (approx. 90%product). The residue was purified on a 3.5 kg silica gel column andeluted using EtOAc/Hexane(4:1). Pure product fractions were evaporatedto yield 96 g (84%).

3'-O-Acetyl-2'-O-Methoxyethyl-5'-O-dimethoxytrityl-5-methyl-4-triazoleuridine:

A first solution was prepared by dissolving3'-O-acetyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methyluridine (96g, 0.144 M) in CH₃ CN (700 mL) and set aside. Triethylamine (189 mL,1.44 M) was added to a solution of triazole (90 g, 1.3 M) in CH₃ CN (1L), cooled to -5° C. and stirred for 0.5 h using an overhead stirrer.POCl₃ was added dropwise, over a 30 minute period, to the stirredsolution maintained at 0-10° C., and the resulting mixture stirred foran additional 2 hours. The first solution was added dropwise, over a 45minute period, to the later solution. The resulting reaction mixture wasstored overnight in a cold room. Salts were filtered from the reactionmixture and the solution was evaporated. The residue was dissolved inEtOAc (1 L) and the insoluble solids were removed by filtration. Thefiltrate was washed with 1×300 mL of NaHCO₃ and 2×300 mL of saturatedNaCl, dried over sodium sulfate and evaporated. The residue wastriturated with EtOAc to give the title compound.

2'-O-Methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine:

A solution of3'-O-acetyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methyl-4-triazoleuridine(103 g, 0.141 M) in dioxane (500 mL) and NH₄ OH (30 mL) was stirred atroom temperature for 2 hours. The dioxane solution was evaporated andthe residue azeotroped with MeOH (2×200 mL). The residue was dissolvedin MeOH (300 mL) and transferred to a 2 liter stainless steel pressurevessel. MeOH (400 mL) saturated with NH₃ gas was added and the vesselheated to 100° C. for 2 hours (tlc showed complete conversion). Thevessel contents were evaporated to dryness and the residue was dissolvedin EtOAc (500 mL) and washed once with saturated NaCl (200 mL). Theorganics were dried over sodium sulfate and the solvent was evaporatedto give 85 g (95%) of the title compound.

N⁴ -Benzoyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine:

2'-O-Methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine (85 g, 0.134 M)was dissolved in DMF (800 mL) and benzoic anhydride (37.2 g, 0.165 M)was added with stirring. After stirring for 3 hours, tlc showed thereaction to be approximately 95% complete. The solvent was evaporatedand the residue azeotroped with MeOH (200 mL). The residue was dissolvedin CHCl₃ (700 mL) and extracted with saturated NaHCO₃ (2×300 mL) andsaturated NaCl (2×300 mL), dried over MgSO₄ and evaporated to give aresidue (96 g). The residue was chromatographed on a 1.5 kg silicacolumn using EtOAc/Hexane (1:1) containing 0.5% Et₃ NH as the elutingsolvent. The pure product fractions were evaporated to give 90 g (90%)of the title compound.

N⁴-Benzoyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine-3'-amidite:

N⁴ -Benzoyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine (74g, 0.10 M) was dissolved in CH₂ Cl₂ (1 L) Tetrazole diisopropylamine(7.1 g) and 2-cyanoethoxy-tetra-(isopropyl)phosphite (40.5 mL, 0.123 M)were added with stirring, under a nitrogen atmosphere. The resultingmixture was stirred for 20 hours at room temperature (tlc showed thereaction to be 95% complete). The reaction mixture was extracted withsaturated NaHCO₃ (1×300 mL) and saturated NaCl (3×300 mL). The aqueouswashes were back-extracted with CH₂ Cl₂ (300 mL), and the extracts werecombined, dried over MgSO₄ and concentrated. The residue obtained waschromatographed on a 1.5 kg silica column using EtOAc/Hexane (3:1) asthe eluting solvent. The pure fractions were combined to give 90.6 g(87%) of the title compound.

Example 2

Cell culture and treatment with phorbol esters and oligonucleotides

PKC protein half-lives have been reported to vary from 6.7 hours to over24 hours (Young et al., Biochem. J. 244:775-779 (1987); Ballester etal., J. Biol. Chem. 260:15194-15199 (1985)). These long half-lives makeinhibiting steady-state levels of PKC-α an unwieldy approach whenscreening antisense oligonucleotides, due to the long incubation timeswhich would be required. We have therefore made use of the ability ofphorbol esters to reversibly lower intracellular levels of PKC.Treatment of cells with phorbol esters causes an initial activation ofkinase activity, followed by a down-regulation of PKC. For PKC-α thisdown-regulation has been shown to be a direct consequence of anincreased rate of proteolysis of the kinase with no apparent change insynthetic rate.

We determined that in human lung carcinoma (A549) cells, treatment withthe phorbol ester 12,13-dibutyrate (PDBu), using a modification of themethod of Krug et al., (Krug et al., J. Biol. Chem. 262:11852-11856(1987)) lowered cellular levels of PKC-α, without affecting PKC-α mRNAlevels, and that this effect was reversible. The basis of the assay toscreen for potency of oligonucleotides targeting PKC-α is to initiallylower PKC-α protein levels by chronic treatment with PDBu, remove PDBuby extensively washing the cells (hence allowing the cells to synthesizefresh PKC-α protein), and incubate the cells with oligonucleotidesintended to inhibit the resynthesis of new PKC-α protein.

Procedure: A549 cells (obtained from the American Type CultureCollection, Bethesda Md.) were grown to confluence in 6-well plates(Falcon Labware, Lincoln Park, N.J.) in Dulbecco's modified Eagle'smedium (DME) containing 1 g glucose/liter and 10% fetal calf serum (FCS,Irvine Scientific, Santa Ana, Calif.).

Cells were treated with 500 nM PDBu (Sigma Chem. Co., St. Louis, Mo.)for 12-16 hours (overnight). Cells were then washed three times in DMEat 37° C., and 1 ml DMA containing 20 μl DOTMA (Lipofectin reagent, BRL,Bethesda, Md.) was added. Oligonucleotides were added to a concentrationof 1 μM and the cells were incubated for a further 4 hours at 37° C.

Cells were washed once in 3 ml DME containing 0.1 mg/ml BSA and afurther 2 ml DME containing 0.1 mg/ml BSA was added. Oligonucleotides (1μM) were added and the cells were incubated at 37° C. for 24 hours.

Cells were washed three times in phosphate-buffered saline (PBS) andcellular proteins were extracted in 120 μl sample buffer (60 mM Tris pH6.8, 2% SDS, 10% glycerol, 10 mM dithiothreitol) and boiled for 5minutes. Intracellular levels of PKC-α protein were determined byimmunoblotting.

Example 3

Effect of ISIS 3521 on PKC protein expression

Cell extracts were electrophoresed on 10% SDS-PAGE mini-gels. Theresolved proteins were transferred to Immobilon-P membrane (Millipore,Bedford Mass.) by electrophoretic transfer and the membrane was blockedfor 60 minutes in TBS (Tris-HCl pH 7.4, 150 mM NaCl) containing 5%nonfat milk. The membrane was then incubated for 16 hours at 4° C. withmonoclonal antibodies raised against PKC-α (UBI, Lake Placid N.Y.)diluted to 0.2 μg/ml in TBS containing 0.2% nonfat milk. This wasfollowed by three washes in TBS plus 0.2% nonfat milk. The membrane wasthen incubated for one hour with ¹²⁵ I-labelled goat anti-mousesecondary antibody (ICN Radiochemicals, Irvine Calif.). Membranes werethen washed extensively in TBS plus 0.2% nonfat milk. Bands werevisualized and quantitated using a Phosphorimager (Molecular Dynamics,Sunnyvale, Calif.). PKC-α appears as a single band with a molecularweight of 80 kD.

Each oligonucleotide was tested three times, in triplicate, and theresults of the experiments were normalized against percentage of proteinpresent as compared to cells which were not treated witholigonucleotide. Oligonucleotide ISIS 3521 (5'-GTTCTCGCTGGTGAGTTTCA, SEQID NO: 1), targeted to the 3' untranslated region of PKCα, reduced PKCprotein levels by approximately 48% compared to untreated controls.

Example 4

Effect of oligonucleotides having SEQ ID NO: 1 on PKC-α mRNA levels

A549 cells were treated with phosphorothioate oligonucleotides at 500 nMfor four hours in the presence of the cationic lipids DOTMA/DOPE, washedand allowed to recover for an additional 20 hours. Total RNA wasextracted and 20 μg of each was resolved on 1.2% gels and transferred tonylon membranes. These blots were probed with a ³² P radiolabeled PKC-αcDNA probe and then stripped and reprobed with a radiolabeled G3PDHprobe to confirm equal RNA loading. Each oligonucleotide (3520, 3521,3522 and 3527) was used in duplicate. The two major PKC-α transcripts(8.5 kb and 4.0 kb) were examined and quantified with a PhosphorImager(Molecular Dynamics, Sunnyvale Calif.). ISIS 3521 (SEQ ID NO: 1) gaveapproximately 80% reduction of the smaller transcript and over 90%reduction of the larger transcript.

Two oligonucleotides having SEQ ID NO: 1 and an 8-deoxynucleotidecentral region flanked on each side by nucleotides having the 2'-O--CH₂CH₂ OCH₃ modification were synthesized. For ease of synthesis, the lastnucleotide was a deoxynucleotide. These compounds, shown in Table 1,differ in that one of them, ISIS 9606, has a uniform phosphorothioatebackbone while the other, ISIS 9605, has a phosphorothioate backbone inthe central region (backbone linkages 7-14) and a phosphodiesterbackbone in the remaining (flanking) regions. These oligonucleotideswere tested for their ability to inhibit PKCα mRNA expression in A549cells, in comparison to the phosphorothioate compound, ISIS 3521. Theresults are shown in FIGS. 1A and 1B. IC50s were calculated(oligonucleotide concentration yielding 50% inhibition) for the threecompounds. The phosphorothioate compound, ISIS 3521, showed an IC50 ofapproximately 170 nM. Both the methoxyethoxy compounds, ISIS 9605 and9606, showed IC50s of approximately 25 nM. This 6-to-7- fold increase inpotency with the methoxyethoxy modification was an indication ofsurprising activity. Because of their extremely low IC50s, the2'-methoxyethoxy compounds 9605 and 9606 are preferred.

                  TABLE 1                                                         ______________________________________                                        Oligonucleotides having SEQ ID NO: 1                                          ISIS #                                                                        ______________________________________                                        3521     GsTsTsCsTsCsGsCsTsGsGsTsGsAsGsTsTsTsCsA                              9605     GoToToCoToCsGsCsTsGsGsTsGsAsGoToToToCoA                              9606     GsTsTsCsTsCsGsCsTsGsGsTsGsAsGsTsTsTsCsA                              12723    GoToToCoToCsGsCsTsGsGsTsGsAsGoToToToCoA                              ______________________________________                                         bold = 2O--CH.sub.2 CH.sub.2 OCH.sub.3                                        s = phosphorothioate (P═S) linkage                                        o = phosphodiester (P═O) linkage                                     

Example 5

Culture of human A549 lung tumor cells

The human lung carcinoma cell line A549 was obtained from the AmericanType Culture Collection (Bethesda Md.). Cells were grown in Dulbecco'sModified Eagle's Medium (Irvine Scientific, Irvine Calif.) containing 1gm glucose/liter and 10% fetal calf serum (Irvine Scientific). Cellswere trypsinized and washed and resuspended in the same medium forintroduction into mice.

Example 6

Effect of ISIS 3521 on the growth of human A549 lung tumor cells in nudemice

200 μl of A549 cells (5×10⁶ cells) were implanted subcutaneously in theinner thigh of nude mice. ISIS 3521, a phosphorothioate oligonucleotidewith SEQ ID NO: 1, was administered twice weekly for four weeks,beginning one week following tumor cell inoculation. Oligonucleotideswere formulated with cationic lipids (DMRIE/DOPE) and givensubcutaneously in the vicinity of the tumor. Oligonucleotide dosage was5 mg/kg with 60 mg/kg cationic lipid. Tumor size was recorded weekly.

As shown in FIG. 2, tumor growth was almost completely inhibited in twoof the three mice, and reduced compared to control levels in the thirdmouse. This inhibition of tumor growth by ISIS 3521 is statisticallysignificant. The control oligonucleotide (ISIS 1082) is a 21-merphosphorothioate oligonucleotide without significant sequence homologyto the PKC mRNA target.

Administration of oligonucleotides to mice whose tumors had alreadyreached detectable size had no discernable effect on subsequent tumorgrowth.

Example 7

Effect of ISIS 3521 on growth of human MDA-MB231 tumors in nude mice

MDA-MB231 human breast carcinoma cells were obtained from the AmericanType Culture Collection (Bethesda, Md.). Serially transplanted MDA-MB231tumors were established subcutaneously in nude mice. Beginning two weekslater, ISIS 3521 was administered intravenously, in saline, daily for 14days at dosages of 60 mg/kg and 6 mg/kg. Control oligonucleotide ISIS1082 was also administered at these doses, and a saline control was alsogiven. Tumor growth rates were monitored for the two-week period ofoligonucleotide administration. As shown in FIG. 3, ISIS 3521significantly inhibited tumor growth at dosages of 60 mg/kg and 6 mg/kg.The control oligonucleotide (ISIS 1082) also showed some reduction intumor growth, but this effect was less than with the antisenseoligonucleotide even at high doses, and considerably less at the lowerdose. A lower-dose study was conducted at 6 mg/kg and 0.6 mg/kg. At 0.6mg/kg ISIS 3521 significantly reduced tumor growth.

Example 8

Effect of ISIS 3521 on the growth of murine Lewis lung carcinoma in mice

Serially transplanted murine Lewis lung carcinomas were established inmice. Oligonucleotides 3521 was administered intravenously every day for14 days at doses of 6 mg/kg and 0.6 mg/kg. Tumor growth rates weremonitored for the two-week period of oligonucleotide administration. Asexpected, this oligonucleotide, targeted to a human PKC sequences, hadinsignificant effects on the mouse-derived tumors.

Example 9

Effects of antisense oligonucleotide ISIS 4189 on endogenous PKC-αexpression in hairless mice

In order to study oligonucleotide effects on endogenous PKC mRNA levelsin normal animals, it was necessary to employ an oligonucleotidecomplementary to the murine PKC-α. ISIS 4189 is a 20-merphosphorothioate oligonucleotide targeted to the AUG codon of mousePKC-α. This region is without homology to the human PKC sequence and theoligonucleotide has no effect on expression of PKC-α in human cells.ISIS 4189 has an IC50 of 200 nM for mRNA reduction in C127 mouse breastepithelial cells. ISIS 4189 in saline was administered intraperitoneallyto hairless mice at concentrations of 1, 10 or 100 mg/kg body weight.Injections were given daily for seven days. Tissues from liver, kidney,spleen, lung and skin were removed and PKC-α mRNA and protein levelswere determined. Histopathological examination was also performed onliver, kidney and lung samples. ISIS 4189 at 100 mg/kg inhibitedendogenous PKC-α mRNA levels in the mouse liver to 10-15% of control(saline) levels.

Example 10

Effect of ISIS 3521 on the growth of human T24 bladder tumors in nudemice

Subcutaneous human T24 bladder carcinoma xenografts in nude mice wereestablished by injection of 5×10⁶ T24 cells under the skin. Mice weretreated with ISIS 3521 or ISIS 4559, a phosphorothioate scrambledversion of the ISIS 3521 sequence, or ISIS 1082, an unrelated controlphosphorothioate oligonucleotide targeted to Herpes simplex virus(oligonucleotide doses 0.006 mg/kg, 0.06 mg/kg, 0.6 mg/kg or 6.0 mg/kg)or saline administered intraperitoneally three times weekly. By day 21,neither ISIS 1082 nor ISIS 4559 had any effect on tumor growth at anydose. By day 21, ISIS 3521 showed a dose-dependent inhibition of tumorgrowth at all dose levels, with a maximal inhibition of 90% at the 6mg/kg dose.

Example 11

Effect of ISIS 3521 on the growth of human Colo-205 colon tumors in nudemice

Subcutaneous human Colo-205 colon carcinoma xenografts in nude mice wereestablished by injection of 5×10⁶ Colo-205 cells under the skin. Micewere treated with ISIS 3521 and an unrelated control phosphorothioateoligonucleotide (ISIS 1082) administered intravenously once per day at adosage of 6.0 mg/kg. In this study, ISIS 3521 inhibited tumor growthafter 25 days by 84% compared to saline controls. The controloligonucleotide, ISIS 1082, inhibited tumor growth by 20%.

Example 12

Effect of ISIS 3521 on U-87 human glioblastoma subcutaneous xenograftsinto nude mice

The U-87 human glioblastoma cell line was obtained from the ATCC(Rockville Md.) and maintained in Iscove's DMEM medium supplemented withheat-inactivated 10% fetal calf serum. Nude mice were injectedsubcutaneously with 2×10⁷ cells. Mice were injected intraperitoneallywith ISIS 3521 at dosages of either 2 mg/kg or 20 mg/kg for 21consecutive days beginning 7 days after xenografts were implanted. Tumorvolumes were measured on days 14, 21, 24, 31 and 35. On day 35 (7 daysafter end of treatment), ISIS 3521 at 2 mg/kg had reduced tumor volumeby 84% compared to saline or sense oligonucleotide control. The 20 mg/kgdose reduced tumor size by 91% on day 35.

Example 13

Effect of ISIS 3521 on PKC-α protein levels in U-87 glioblastomaxenograft in nude mice

PKCα protein levels in subcutaneous U-87 tumor xenografts were measuredby Western blot analysis on day 24 (day 17 of treatment with ISIS 3521)and day 35 (7 days after end of treatment with ISIS 3521). Anaffinity-purified PKCα-specific polyclonal antibody (Life Technologies,Inc.) was used as the primary antibody. By day 24, ISIS 3521 was foundto virtually totally abolish PKCα in the tumors. By seven days aftercessation of oligonucleotide treatment (day 35), PKCα had returned tocontrol levels.

Example 14

"Crossover experiment" to evaluate effect of switching treatment ontumor size

The two groups of mice with subcutaneous U-87 xenografts previouslytreated with ISIS 3521 (2 mg/kg or 20 mg/kg) were switched to differenttreatments on day 35 (7 days after the initial 21 day treatment hadended). The group which had previously received 20 mg/kg ISIS 3521 nowreceived saline ("high dose-to-control"). The group which had received 2mg/kg ISIS 3521 now received 20 mg/kg ISIS 3521 ("low dose-to-highdose"). This crossover treatment was continued for 21 days as for theoriginal treatment. As shown in FIG. 4, the growth of the tumors in the"low dose-to-high dose" group (open triangles) leveled off aftertreatment was switched (arrow). The growth of the tumors in the "highdose-to-control" group (closed triangles) rapidly accelerated afterswitching to saline treatment (arrow). S=sense oligonucleotide(control); AS=antisense oligonucleotide (ISIS 3521) targeted to PKCα.

Example 15

Effect of ISIS 3521 on intracerebral U-87 glioblastoma xenografts intonude mice

U-87 cells were implanted in the brains of nude mice. Mice were treatedvia continuous intraperitoneal administration of antisenseoligonucleotide ISIS 3521 (20 mg/kg), control sense oligonucleotide (20mg/kg) or saline beginning on day 7 after xenograft implantation. Allmice survived until day 25, at which point the saline-treated mice beganto die. All saline-treated mice and sense oligonucleotide-treated micewere dead by day 41. In contrast, all ISIS 3521-treated mice were aliveuntil approximately day 37, and half of the mice were still alive at day61. At the termination of the experiment at day 80, 40% of the ISIS3521-treated mice were still alive.

Example 16

Effect of the 2'-methoxyethoxy oligonucleotide ISIS 12723 on the growthof human Colo-205 colon tumors in nude mice

Subcutaneous human Colo-205 colon carcinoma xenografts in nude mice wereestablished by injection of 5×10⁶ Colo-205 cells under the skin. Micewere treated with ISIS 12723 (SEQ ID NO: 1 with an 8-deoxynucleotidecentral region flanked on each side by six nucleotides having the2'-O-CH₂ CH₂ OCH₃ modification, a phosphorothioate backbone in thecentral region (backbone linkages 7-14) and a phosphodiester backbone inthe remaining (flanking) regions) or ISIS 3521 (SEQ ID NO: 1, fullydeoxy phosphorothioate), administered intravenously once per day at adosage of 0.006, 0.06, 0.6 or 6.0 mg/kg. As shown in FIG. 5, in thisstudy, ISIS 12723 inhibited tumor growth by over 95% compared to salineplacebo controls. ISIS 3521 inhibited tumor growth by over 83% comparedto controls. The methoxyethoxy compound, ISIS 12723, is thereforepreferred.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 1                                             - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 # 20               TTCA                                                       __________________________________________________________________________

What is claimed is:
 1. An oligonucleotide comprising SEQ ID NO: 1,wherein at least one nucleotide of said oligonucleotide comprises a2'-O--CH₂ CH₂ OCH₃ modification of its sugar moiety.
 2. Theoligonucleotide of claim 1, wherein the intersugar linkages between eachof the nucleotides of SEQ ID NO: 1 are phosphorothioate linkages.
 3. Theoligonucleotide of claim 1, wherein the intersugar linkages between eachof the nucleotides of SEQ ID NO: 1 are phosphodiester linkages.
 4. Theoligonucleotide of claim 1, wherein the intersugar linkages between eachof the nucleotides of SEQ ID NO:1 are a mixture of phosphodiesterlinkages and phosphorothioate linkages.
 5. A method of inhibiting PKCexpression in cells comprising contacting the cells with anoligonucleotide of claim
 1. 6. The method of claim 5 wherein the cellsare cancer cells.
 7. A method of treating a condition associated withexpression of PKC comprising administering to an animal, cells, tissues,or a bodily fluid thereof, a therapeutically effective amount of anoligonucleotide of claim
 1. 8. The method of claim 7 wherein saidcondition is an inflammatory or hyperproliferative disorder.
 9. Themethod of claim 8 wherein the condition is cancer or psoriasis.